Contaminant Detection in Soybean Using Allele-specific Digital PCR on the TaqMan® OpenArray® Platform

Contamination of seed lots by unexpected varieties is a problem in quality assurance of certified seeds. Identification and elimination of contaminated seed lots are essential to maintain the quality of seed stocks. Cost efficient and high-throughput methods for identifying low contamination level are needed. We are presenting a feasibility study for contaminant detection in soybean using digital PCR on the TaqMan® OpenArray® platform. Digital PCR is a sensitive approach to detect rare alleles and allows absolute quantification without using endogenous controls. Combining digital PCR with the TaqMan® OpenArray® system enables high-throughput contaminant screening. Six SNPs were selected to distinguish soybean variety A from the contaminant variety B.  Allele specific Custom TaqMan® Assays were designed for the OpenArray® digital PCR. A spike-in experiment was set up mixing soybean variety A DNA with contaminant variety B DNA at a 10,000:1 ratio to mimic 0.01% contamination in the seed lot. Digital PCR was performed on the OpenArray® PCR instrument using real-time signal acquisition followed by data analysis for quantification of detected copies/µl of samples. Specificity of the assays in discriminating contaminant in a background of high concentration of variety A DNA was tested. All contaminant-specific assays produced no false-positive results in variety A DNA background and achieved the targeted sensitivity of detecting 0.01% contamination. This methodology combining digital PCR with the high-throughput OpenArray® platform is applicable to any type of rare allele detection like contaminant detection or identifying genetically modified plant seeds in a pool of wild-type seeds. For Research Use Only.  Not intended for animal or human therapeutic or diagnostic use.